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Chavterl %GENERAL INTRODUCTION
General Introduction
Ocean, the vast and the least understood ecosystem, its relentless and
impredictable moods, the myriad life forms above and below its restless surface,
the vast treasure of energy sources hidden under the deep trenches, have all
fascinated and lured man. The potent resources of the oceans that lie unexplored
and unexploited perceptibly influence the health and wealth of a nation.
The oceans are unique sources of diverse array of natural products. Apart
from viewing oceans as the avenue for cheap source of protein, other usage like
the pharmaceutical importance for producing drugs against many vagaries sounds
encouraging. It is increasingly recognized that a huge number of natural products
and novel chemicals entities exist in the oceans, with biological activities that may
be useful in the quest for drugs with greater efficacy and specificity for the
treatment of many human diseases.
The marine environment is an exceptional reservoir of bioactive natural
products, many of which exhibit structural/chemical features not found in terrestrial
natural products. Marine animals fight daily for both food and survival and this
underwater warfare is waged with chemicals which are potent sources of many
therapeutic compounds. Biomolecules extracted from the aquatic organisms have
been proved to have powerful bioactive and biomedical properties. Attempts are
being made to develop drugs from the sea which have the potential asanticarcinogenic, antibiotic, growth regulatory, haemolytic, haemagglutinating,
analgesic, antispasmodic, hypotensive or hypertensive agents, and even anti-HIV
agents. To date approximately 16,000 marine natural products have been isolated
from marine organisms and reported in approximately 6,800 publications (Bhakuni
and Rawat 2005).
2
General Introduction
Biotoxins from marine organisms usually come to the attention of biologists
because they exert a striking effect on the organisms in marine community. Many
sea creatures can directly injure, envenom, and poison their victims. Biotoxins are
found throughout the entire phylogenetic series of marine animals. There is
substantial practical motivation for studying marine biotoxins, for like other toxins
they are highly active in biological sense and therefore useful in biomedical
research. Chemically the toxins make the most challenging targets because of
their structural complexity and extremely limited availability. To pharmacologists
and biochemists marine toxins are attractive because of their potent and specificacfion.
Marine toxins have a wide range of biological activities and often these
activities are induced by minute amounts of toxin, i.e., by nature toxins are highly
potent molecules. The biological activities exhibited by the toxins are harmful to
the target organisms, since the function of the toxin is either to protect the toxic
species from attack by a predator or to immobilize potential prey. Despite the
general harmful effects in the target organisms, toxins have a great potential to be
harnessed for favorable effect on non-target organisms and in particular humans
for therapeutic purposes. Biological significance of marine biotoxins is rather an
important subject in medical science. The scientific importance of biotoxins is
frequently misunderstood, because the most popular attitude among the public is
that poisons are lethal substances, causing intoxications and death and are
therefore substances to be avoided. Research activities on biotoxins are justified
because of the fact that knowledge on their pharmacological and chemical
properties can be useful in developing an antitoxin or decephering their clinicalcharacteristics.
The perspective science of Biotoxinology imbibes to have absoluteknowledge of any organism by solving a number of complex biological and
3
General Introduction
biochemical conundrums yielding in several tangible results in terms of human
health security and economic benefits. Natural toxins are unique toxins, which
possess some common properties whether they are obtained from plants,microorganisms, or animals. One common characteristic is that they exert a
prominent effect on the metabolism and biological functions of the intoxicated
animals with just a minute quantity.
Biotoxins are mainly of two types; Phytotoxins or plant poisons andZootoxins or animal poisons. A large variety and number of marine creatures
inhabiting the ocean waters pose threat to humans by virtue of their ability either to
bite, lacerate or sting or contain toxic substances within their flesh, blood etc. ln
the past several decades the research carried out in the field of Fish Biotoxinology
has acquired new dimensions. The types of toxic fish, the nature and source of
toxins and the conditions governing their toxicity are all topics of interest in this
field. The information regarding the source of adventitious toxicity of fish is not only
vital for development of antidotes and rational assessment of the usefulness of the
fish species as food source, but it also opens an arena for the exploration of new
biologically active chemical substances or biodynamic compounds of therapeuticvalue.
Halstead and Courville (1970), Concon (1988), and Halstead (2001) have
classified ichthyotoxic fish into two groups: a) Poisonous or Phenerotoxic fish
which when ingested cause a biotoxication in humans due to a toxic substance
present in the fish and b) Venomous or Acanthotoxic fish that produce poison by
means of glandular structures that are equipped with a traumatogenio device to
purvey their venoms. Intermediate to the poisonous and venomous fish are the
crinotoxic fish that produce a poison by means of glandular structures independent
of true venom apparatus.
4
General Introduction
Poisonous Fish: Research in the past several decades has increased our
knowledge on the types of toxic fish, the nature and source of toxins and the
conditions governing their toxicity. The major problem facing the marine
biotoxinologist is the variability and frequent unpredictability of the toxicity of this
segment of marine life. The degree of toxicity of these fish may fluctuateperiodically. The poisonous substances in fish may be concentrated in specific
tissues or organs. Based on the tissue in which the toxin is present in the fish the
poisonous fish are further classified into lchthyosarcotoxic (flesh, musculature,viscera or skin) eg: herrings, anchovies, tarpons and pufferfish, lchthyootoxic (roe
or gonad) eg: The most dangerous forms are the genera Barbus, Schizothorax,
Tinca (Cyprinidae) and Stichaeus (Stichaeidae). lchthyohaemotoxic (blood or
serum) eg: Those fish having poisonous blood, members of the anguilliform
families Anguillidae, Congridae, Muraenidae, and Ophichthidae lchthyohepatotoxic
(liver) eg: sharks
Crinotoxic fish: Crinotoxic fish are intermediate to poisonous andvenomous fish and include filefish, pufferfish, trunkfish, boxfish, toadfish, gobyfish,
catfish etc. They are able to secrete substances from their skin, known asicthyocrinotoxins, that are capable of repelling or incapacitating other marine
animals (Klaassen and Watkins, 1999). These secretions are also thought to
possess antibiotic activity, protecting fish from the myriad of invadingmicroorganisms in the marine environment, a fact that has prompted recent
research on them. These have specialized cells or glands in their skin but lack a
parenteral mechanism (Perriere and Perriere 2003). Glandular secretions are
normally released into the surrounding medium, the water. Crinotoxins are often
called mucus toxins. The poison glands of ichthyocrinotoxic fish assist in the
defensive mechanism of the fish by producing warning or repellent substances
especially under stress conditions (Deo, 2000).
5
General Introduction
Venomous fish: Fish constitute almost half the number of vertebrates on
earth (Nelson, 1984), and approximately 22,000 species of fish are contained in
some 50 orders and 445 families. Of these, nearly 1,200 species of marine fish,
including stingrays, scorpionfish, zebrafish, stonefish, weeverfish, toadfish,
stargazers, and some species of shark, ratfish, catfish, surgeonfish and blenny,
are known or suspected to be venomous (Smith and Wheeler, 2006). The vast
majority of these fish is non-migratory, slow moving, and tend to live in shallow
waters in protected habitats (Maretic, 1988). It has been suggested that this
tendency towards inactivity is closely linked with the evolution of venom apparatus
(Cameron and Endean, 1973).
Although only a handful of species of venomous fish are thought to be
capable of causing human mortality, many other species of fish can produce
severe envenomation. While not considered life threatening, envenomation by
these fish is associated with considerable pain, and their venoms contain many
pharmacologically active components. Therefore, these species are important as
sources of pharmacological compounds that may be useful as research tools or
lead compounds for drugs.
Venomous or Acanthotoxic fish produce their poison by means of glandular
structure and are equipped with a traumatogenic device (teeth, spine, sting, etc.)
to purvey their venom (Church and Hodgson, 2002). The venom apparatus
invariably consists of spines that may be located on the dorsal, pectoral, opercular,
shoulder, pelvic, anal and caudal areas of the fish depending on the species
(Williamson, 1995). Venom is delivered when the spine pierces the tissue of the
victim, the integumentary sheath enclosing the spine and venom is ruptured, and
the venom enters the wound (Church and Hodgson 2002).
It is thought that venom apparatus evolved relatively recently in the
development of these fish, because fish despite being on a higher level of
6
General Introduction
development than some other groups of venomous creatures (e.g. spiders), their
venom apparatus are much more primitive, relying on a completely involuntary
mechanical action, rather than a voluntary expulsion of venom (Maretic, 1988).
Venomous spines can be observed in species from many evolutionary classes of
fish, from the primitive cartilaginous fish like stingrays, to the more advanced bonyfish such as stonefish.
As venoms from various animals including fish have a wide range ofpharmacological effects on human nervous, muscular, and cardiovascular
systems, the venom proteins offer a source for the development of drugs for the
treatment of pain, cancer, infectious diseases, auto-immune diseases, allergies,
and hypertension. Most pharmaceutical drugs have been derived from natural
products (compounds discovered in nature). However, most organisms, including
fish, have only recently been examined for biologically active molecules (or
compounds that affect the biochemistry of living things) that could be developed
into potential drugs. ln addition, pharmaceutical companies have recently de
emphasized natural products research and search for biologically active molecules
by screening mass produced combinations of compounds. Neither strategy has
met expectations, and the number of new registered drugs continues to decrease
dramatically. To date, most venom bioprospecting has focused on snakes,
resulting in six-stroke or cancer treatment drugs that are nearing U.S. Food and
Drug Administration review (Smith and Wheeler, 2006). However, as fish, not
snakes, are now known to be the dominant venomous group among vertebrates,
they represent a massive untapped resource of medically beneficial compounds.
Studies on Acanthotoxic fish from the subcontinent of India are very scarce.
The present investigation aims at elucidating the bioactivities of the spine extract
of Scatophagus argus (Family: Scatophagidae) from Cochin estuary that would
qualify them as a potential source of biopharmaceutical compounds. Injuries
7
General Introduction
inflicted by the fish cause pain disproportionate to the resultant wound even in the
absence of notable secondary tissue injury. Secondly the quality and duration of
the pain caused by the venom and its and heat labile nature suggest potential
soluble protein-venom-nocicepter interaction. The study of biological properties
and chemistry of the venom would make an impact in developing newdetoxification techniques. In the light of the above the main objectives of the
present study are:
> To elucidate a method for extraction of the venom.
> To find out the toxicity of the spine extract of Scatophagus argus in mice.
> To analyze the gross anatomical changes and histopathological changes
caused by the toxin in mice models.
‘> To elucidate the effect of the S. argus venom on the antioxidant enzymes,
antioxidants, detoxifying enzymes in mice tissues.
> To assess the impact of S. argus venom on cell line.
> To study the neuromuscular toxicity of the S. argus venom.
> To find out the biomedical application of the S. argus venom.
> To understand the lysosomal membrane stability against the S. argusvenom.
> To characterize the toxin and to assay the different enzymes andbiochemical molecules present in the S. argus venom.
8
General Introduction
S CA TOPHA GUS ARGUS
Scatophagus argus belonging to the family Scatophagidae are widely
distributed in brackish water and marine habitats of lndo-Pacific, the Malay
Archipelago, the Philippines, Australia, South and Southeast Asia especially India
where they inhabit estuaries, coastal mud flats, mangrove swamps, harbors and
upstream swamps. They are commonly known as butterfish, argus fish, spade
fish, spotted spade fish [Barry and Fast, 1988], leopard pomfret [Mookerjee et aI.,
1949] and spotted scat [Bardach et aI., 1972]. The word Scatophagus means "offal
eater’ or “eater of feces” and the word argus means “thousand eyed” which refers
to the spot found on all juvenile and larger fish. Scats are well adapted to live in
ever changing environment that endows them with many biological attributes
highly desired in cultured finfish. The euryhaline nature and the beautifully spotted
rhombic body rank it as a fascinating aquarium fish while the quality and taste
ranks it as a highly priced edible fish.
Phylum
Class
Subclass
lnfraclass
Super Order
Order
Suborder
Family
Genera
Species
Chordata
Osteichthyes
Neopte rygii
Teleostei
Aca nthopterygii
Perciformes
Acanthuroidei
Scatophagidae
Scatophag us
argus
Scats are greenish brown in colour with dull white belly and black blotches
of varying sizes all over the body. Body is rhombic and well compressed with firm
skin and small ctenoid scales. Rostrodorsal profile strongly ascending and
followed by a deep concavity above the eyes. Mouth is small, terminal, and
9
General Introduction
horizontal with bands of fine teeth in jaws but not on palate. It is generallybelieved that the fish spawn in the neighbourhood of coral reefs but the young
migrate to the fresh water at the mouth of rivers and estuaries, until they grow
large enough well adapted to live in the sea. (Pic1.1)
Scats are venomous fish and the venom apparatus is constituted by 11
dorsal spines, a pair of ventral spine and 4 anal spines, elongated venom glands
and an integumentary sheath enveloping all [Cameron and Endean, 1977]. The
spines are very sharp and pointed each possessing a pair of antero-lateralgrooves one on each side accommodating the paired venom glands in each spine.
The venom glands are irregularly shaped tending to be columnar in the deepest
portion of the gland. The glands are aggregations of large gland cells in the
thickened epidermis of the integumentary sheath. The venom glands are not
encapsulated in connective tissue sheath. Elongate supporting cells occur among
the venom glands cells some of which possess unusual rod like bodies in their
cytoplasm.
Scats are not aggressive and do not actively attempt to inflict wound.
However they frequently induces accidents in fishermen while handling it. Of the
family Scatophagidae S. argus is said to inflict more painful wounds than do allied
species [Marshall, 1964]. During envenomation the spines are erected and the
mechanical pressure on the spine tears or pushes down the integumentary sheath
over the spine as the venom passes into the wound. Though the puncture is
painful which aches for many hours [Barry and Fast, 1988] it is rarely life
threatening. The butterfish envenoming appears within 5-10 min as excruciating
and persistent local pain disproportionate to the size of injury, redness, swelling
and throbbing sensation that extend to the limbs followed by dizziness. Most
patients are treated with empirical procedure such as emersion of the woundedarea in hot water.
10
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Scatophagus argus
Scatophagus argus male and female